Abstract This project aims to define novel signaling mechanisms for the regulation of innate immune responses by Gasdermins D and E (GSDMD/E) in granulocytic leukocytes that include neutrophils, mast cells, and eosinophils. Physiological roles for GSDMD in both pyroptosis and IL-1? release during inflammasome signaling have been extensively characterized in macrophages and other mononuclear myeloid leukocytes. This involves cleavage of GSDMD by caspase-1 to generate N-GSDMD fragments which oligomerize in the plasma membrane (PM) to form pores that mediate IL-1? efflux and pyroptosis. Notably, the granulocytic myeloid leukocytes are also important sources of IL-1? during multiple innate immune responses, but few studies have examined roles for GSDMD or other GSDM-family proteins in these cells. Our preliminary data shows that caspase-1-generated N-GSDMD in neutrophils does not localize to the PM to form pores or drive pyroptosis but is required for IL-1? secretion. This absence of PM pores reflects alternative subcellular trafficking whereby N-GSDMD associates with azurophilic secretory granules (AG) and LC3+ autophagosomes. Analyses using ATG7-deficient neutrophils indicate that IL-1? is secreted from neutrophils via an autophagy machinery-assisted mechanism. These findings reveal fundamental differences in GSDM biology, including high expression of GSDME, between granulocytes and macrophages that will shape granulocyte roles in innate immunity. We hypothesize that the abundant secretory granules which define neutrophils, mast cells and eosinophils underlie granulocyte-specific mechanisms for non-canonical production of bioactive IL-1? and for regulated cell death via alternative pathways of GSDM family processing and subcellular trafficking. In Aim 1, we will define signaling hierarchies based on activation of inflammatory caspases, granule-derived serine proteases, apoptotic caspases, GSDMD, and GSDME that facilitate alternative paths of bioactive IL-1? production by neutrophils, mast cells, and eosinophils. Studies will include analyses of GSDMD/E trafficking to secretory granules versus the plasma membrane and GSDMD's ability to act as a chaperone for autophagy protein-assisted secretion of proIL-1? that can be extracellularly cleaved by serine proteases. In Aim 2, we will define GSDMD/E signaling networks that regulate granulocyte death pathways which are induced by triggers of extracellular DNA trap release/ETosis, apoptosis, and progression to granulocyte senescence. The project will draw on the strengths of the other three projects to facilitate our functional studies. The results will define novel mechanisms for how GSDMD and GSDME mediate granulocyte-specific signaling responses which play physiological roles during acute infection but contribute to tissue dysfunction in humans with common autoimmune, allergic, or chronic inflammatory diseases.